Several degenerative neural diseases are caused by the improper folding or processing of proteins or by prions, both of which result in invasive neural depositions known as amyloid plaques. The most widely known degenerative neural disease is probably Alzheimer's Disease (AD).
The incidence of AD warrants an urgent and unmet medical need: between 10 and 40% of all people aged 65 to 85 develop AD. Moreover, this segment of the population continues to grow exponentially. Therefore, from a humane, as well as from a social and economical point of view, it is imperative to find ways to efficiently diagnose and treat this devastating disorder. Concerning treatment, drugs are needed not only to slow or stop the disease progression, but also to restore brain damage that has already occurred during the initial stages of AD (before diagnosis). At this moment, neither early-diagnosis nor therapy treatment are efficient.
AD is defined as a dementia that coincides with the presence in the brain of extracellular amyloid plaques, composed mainly of amyloid peptides, and by intracellular neurofibrillary tangles (NFT) composed mainly of protein tau.
A primary component of amyloid plaques characteristic of AD is beta amyloid peptide (A-beta), a highly insoluble peptide 39-43 amino acids (aa) in length that has a strong propensity to adopt beta sheet structures, oligomerize and form protein aggregates. A-beta is produced from the A-beta precursor protein (APP) by two proteolytic events. A beta-secretase activity cleaves APP at the N-terminus of A-beta (“beta-site”) between amino acids Met-671 and Asp-672 (using the numbering of the 770 aa isoform of APP). Cleavage at the beta-site yields a membrane-associated APP fragment of 99 aa (C99). A second site within the transmembrane domain of C99 (“gamma site”) can then be cleaved by a gamma-secretase to release A-beta. APP can alternatively be cleaved within its A-beta region, predominantly at the alpha-secretase cleavage site of APP, to produce a C-terminal APP fragment of 83 aa (C83), which can also be further cleaved by gamma-secretase to produce a small soluble secreted peptide, p3. This pathway reduces the potential accumulation of A-beta.
The intra- and extracellular A-beta adopts a beta-sheet conformation and forms intermediates named ADDL (amyloid derived diffusible ligands) and protofibrils, finally precipitates in the form of amyloid fibrils which assemble into amyloid plaques. In these processes, the more hydrophobic A-beta(1-42) peptide (cf. below) is presumed to serve as a nucleating agent around which the plaques steadily grow.
A number of missense mutations in APP have been implicated in forms of early-onset familial AD. All of these are at or near one of the canonical cleavage sites of APP. Thus, the Swedish double mutation (K670N/M671L) is immediately adjacent to the beta-secretase cleavage site and increases the efficiency of beta-secretase activity, resulting in production of more total A-beta. Any of three mutations at APP residue 717, near the gamma-secretase cleavage site, increases the proportion of a more amyloidogenic 42 aa form of A-beta, also named A-beta(1-42), relative to the more common 40 aa form, A-beta(1-40). Two additional mutations of APP have been described which are close but not adjacent to the alpha-site. A mutation (A692G, A-beta residue 21) in a Flemish family and a mutation (E693Q, A-beta residue 22) in a Dutch family each have been implicated in distinct forms of familial AD. The Flemish mutation, in particular, presents as a syndrome of repetitive intracerebral hemorrhages or as an AD-type dementia. The neuropathological findings include senile plaques in the cortex and hippocampus, and usually multiple amyloid deposits in the walls of cerebral microvessels.
Several years ago, the membrane-associated aspartyl protease, BACE (also called memapsin or Asp2) has been shown to exhibit properties expected of a beta-secretase. This enzyme cleaves APP at its beta-site and between Tyr-10 and Glu-11 of the A-beta region with comparable efficiency. A-beta fragments cleaved at this latter site have been observed in amyloid plaques in AD and in media of APP-transfected HEK293 human embryonic kidney cells. Several groups also observed the presence in the database of an additional aspartyl protease, BACE2 (also called Asp1), a close homologue of BACE (now also referred to as BACE1). BACE2 cleaves APP at its beta-site and more efficiently at sites within the A-beta region of APP, after Phe-19 and Phe-20 of A-beta. These internal A-beta-sites are adjacent to the Flemish APP mutation at residue 21, and this mutation markedly increases the proportion of beta-site cleavage product generated by BACE2. Conservative beta-site mutations of APP that either increase (the Swedish mutation) or inhibit (M671V) beta-secretase activity affect BACE1 and BACE2 activity similarly. BACE2, like BACE1, proteolyzes APP maximally at acidic pH.
Mutations in the APP gene or in the presenilin 1 (PS1) gene (carrying “gamma-secretase” activity) cause early-onset familial AD. Examples of APP mutations are the above-mentioned ‘Swedish’ and ‘London’ (717) mutations located respectively near the beta- and gamma-secretase cleavage sites. These mutations increase the formation of A-beta peptides and especially of A-beta(1-42), and thereby increase the formation of amyloid aggregates and plaques. Whereas initially plaques were believed
to be a major trigger for the development of AD, current studies emphasize the role of protofibrils and ADDL as the major toxic components. It is even conceivable that plaques are a mechanism whereby the neurotoxic peptides are actually rendered biologically inactive.
Further information on neurodegenerative diseases and on the role of A-beta therein can be taken from Wisniewski & Konietzko (2008), Lancet Neurol. 7(9), 805-811, Spires-Jones et al. (2009), Neurobiology of Disease, 213-220, and Lichlen & Mohajeri (2008), Journal of Neurochem. 104, 859-874.
Most current treatments of AD target the acetylcholine deficiency using acetylcholinesterase inhibitors such as donepezil (Aricept®), galantamine (Reminyl®), and rivastigmine (Exelon®) which are registered for the treatment of mild to moderate AD. Donepezil is also approved for severe Dementia Alzheimer's type (DAT) in the U.S.A. and Canada. The acetylcholine deficit reflects the degeneration of cholinergic neurons of the basal forebrain and appears to correlate well with the neuropsychiatric manifestations of the disease. Treatment with acetylcholinesterase inhibitors has some beneficial effects (consistent and significant but modest efficacy on clinical measures of cognition and global function), but cannot cure or stop the progression of the disease, as the etiology of the neurodegeneration is left untreated.
Memantine (Axura®, Namenda®, Ebixa®; Merz Pharmaceuticals) is an NMDA receptor antagonist that showed better outcome in comparison to placebo in the clinical domains cognition, activities of daily living and overall clinical response in AD patients with moderate to severe Alzheimer's disease. Memantine remains a symptomatic therapy that is approved for moderate to severe Alzheimer's disease only. It neither cures nor stops the progression of the disease. A combination of memantine and acetylcholinesterase inhibitors has been shown to have superior efficacy in moderately severe to severe DAT but not in the mild to moderate disease stage.
Some current experimental therapeutic strategies focus on A-beta as a target. There are three major research lines:
a) The development of small molecules (often peptido-mimetics) named beta-sheet breakers, which are designed to interfere with the beta-sheet structure of amyloid peptide aggregates. It has been demonstrated that a stable “beta-sheet breaker”, when administered to a transgenic mouse model of AD, is able to penetrate the blood brain barrier and reduce the number of plaques (Permanne et al. (2002), FASEB J. 16, 860-862). It remains to be demonstrated whether this approach results in cognitive protection and/or restoration.
b) The development of small molecules which inhibit the proteolytic processing of APP into amyloid peptides. Inhibitors of the beta- or gamma-secretase should efficiently block the formation of A-beta and hence protect the brain from neurotoxic effects of amyloid. Effects on already existing brain A-beta burden, such as amyloid plaques which have accumulated over years, are not expected.
c) Passive and active vaccination against A-beta. This research line started with the observation by Schenk et al. (1999), Nature 400, 173-177, that vaccination of transgenic AD mice with A-beta(1-42) prevented the formation of amyloid plaques. In a first experiment, monthly vaccination of young adult mice (age 6 weeks) essentially prevented plaque formation and the concomitant inflammatory reaction in the brain, i.e. absence of amyloid plaques, of astrocytosis and microgliosis. Vaccination starting at a later age, when amyloid plaques were already established, resulted in a partial clearance. Subsequently, it was demonstrated that vaccination with A-beta improved the behavioral and memory deficits as measured in the water maze memory tests. Given the side-effects of vaccination with the entire A-beta, alternative shorter peptides have been designed and used to vaccinate transgenic mice. Clinical trials suggested that the active immunization with A-beta is therapeutically active, as demonstrated by eliciting plaque clearance, attenuating plaque-related pathology, decreasing tau levels and slowing patients' cognitive decline. However, a significant number of patients developed autoimmune meningoencephalitis, caused primarily by the infiltration of autoreactive T lymphocytes into the brain in response to active immunization (Ferrer et al. (2004), Brain Pathol. 14, 11-20; Nicoll et al. (2003), Nat. Med. 9, 448-452; Masliah et al. (2005), Neurology 64, 1553-1562).
As an alternative to active immunization approaches, antibodies directed against A-beta may be administered to a patient. Such passive immunization approach was shown to be successful in reducing brain A-beta burden in transgenic AD mice (DeMattos et al. (2001), Proc. Natl. Acad. Sci. USA 98, 8850-8855). The underlying mechanisms remain open for speculation since it was thought unlikely that antibodies could cross the blood-brain barrier and target the plaques present in brain. The authors therefore suggested that the antibody created an ‘A-beta sink’ in the plasma which titrated A-beta out of the brain. Subsequently, using gelsolin and ganglioside 1, it was demonstrated that any A-beta-binding ligand has the potential to reduce amyloid burden in transgenic AD mice without crossing the blood-brain barrier (Matsuoka et al. (2003) J. Neuroscience 23, 29-33). Short-term (24 hours) passive immunization appeared to restore cognitive deficits of transgenic AD mice even without affecting the total brain amyloid load (Dodart et al. (2002) Nature Neuroscience 5, 452-457). The result would suggest that smaller, still soluble aggregates of A-beta are targeted first by some antibodies, and also that these are the most toxic forms of A-beta. Hence, clearance of proto-fibrillar A-beta could restore memory, at least in transgenic APP-mice.
The humanized anti-A-beta monoclonal antibody bapineuzumab (an analogue of the anti-A-beta mouse antibody known as “3D6”) has meanwhile entered clinical trials. However, the first data reported from Phase II trial showed mixed results: Statistically significant effects on several efficacy endpoints were observed in ApoE4 non-carriers only. Furthermore, bapineuzumab was well tolerated and safe in ApoE4 non-carriers, while in ApoE4 carriers, serious adverse events were more frequently observed in bapineuzumab-treated patients than in the placebo arm. Moreover, vasogenic edema events have been observed. The induction of cerebral microhemorrhages has also been described pre-clinically in transgenic APP mice.
Conventional antibodies (containing an Fc part) used in anti-A-beta passive immunizations are suspected to account for the induction of vasogenic edema or microhemorrhages observed in humans and animal models, which are associated with a targeting of cerebral vascular A-beta deposits (Cerebral amyloid angiopathy) leading to microbleedings via ADCC and/or CDC (Wilcock, D M, Colton, C A, CNS Neurol. Disord. Drug Targets (2009) Vol. 8(1):50-64). Finally, the binding affinity of about 2.5 nM of this antibody, as measured by Biacore, is assumed to be too low to induce an effective “peripheral sink effect”.
Another anti-A-beta antibody, solanezumab (humanized antibody m266; LY-2062430), has also entered clinical testings. The maximal plaque load reduction that could be achieved was published to be about 60%. In addition, specificity of this antibody is limited to soluble A-beta, so that binding of aggregates or plaques cannot be expected.
A third anti-A-beta antibody, ponezumab (PF4360365), only binds to A-beta(x-40) molecules, and not to A-beta(x-42) molecules, the latter being assumed to be the (more) pathogenic A-beta species. Its affinity is even lower than the affinity of bapineuzumab, and the risk of cerebral microhemorrhages can not yet be ruled out, due to its ability to bind to A-beta plaques in blood vessels, combined with a remaining ADCC/CDC activity of its Fc portion.
In summary, the above demonstrates that even if A-beta binding and clearance by (classical) antibodies appears to be an attractive mode-of-action for the development of therapeutical agents for the treatment of e.g. AD, other characteristics and effects of such immunoglobulins which have not yet been fully elucidated, such as the pharmacological implications of their property to bind to certain forms of A-beta, make it far more difficult than one might have initially assumed to find and develop safe and efficient therapeutical antibodies.
Antibody fragments, such as immunoglobulin single variable domain antibodies or VHH domains (as defined below), having specificity for A-beta have also been described in the art: WO2004/44204; WO2006/40153; WO2007/35092; WO2008/122441; and WO2009/04494. Binding characteristics of VHHs synthesized by the present inventors in accordance with the above WO publications were unsatisfactory, for which reason they are not supposed to enter clinical development.
WO09/149,185 discloses so-called DVD constructs, and, inter alia, DVD constructs having A-beta binding specificity. Upon combination of two different anti-A-bata variable domains in such DVD constructs, binding of the parental antibodies was maintained, but no increase in affinity was observed by this combination. Moreover, the disclosed DVD constructs contain an Fc part which is present in “classical” antibodies, so that side effects caused by Fc effector functions, such as complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC), cannot be avoided (cf. above).
Definitive diagnosis of AD still requires post-mortem pathological examination of the brain to demonstrate the presence of amyloid plaques, neurofibrillary tangles, synaptic loss and neuronal degeneration. This is essentially the same procedure as defined by Alois Alzheimer in 1906. In 1984 the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) established formal criteria for the diagnosis of AD (reviewed in Petrella et al. (2003), Radiology 226, 315-336). Patients meeting all of the following criteria are diagnosed probable AD: dementia evidenced by examination and testing (e.g. Mini-Mental Test, Blessed Dementia Scale, or similar tests), impairment of memory and at least one other cognitive function, normal consciousness, onset between 40 and 90 years of age, absence of signs of other diseases that cause dementia (exclusion criterion). A gradual progressive, cognitive impairment without an identifiable cause will be diagnosed as possible AD. Probable AD is further defined as mild (early), moderate (middle) or severe (late) dementia. Laboratory analysis is used to objectively define or exclude alternative causes of dementia. ELISA assays of A-beta(1-42) and phospho-tau in cerebrospinal fluid (CSF), combined with genotyping for ApoE4 (a predisposing genetic factor) appear to be sensitive and specific. The methods are, however, not widely applicable because of the invasive CSF puncture, preventing this to become routine screening. ELISA for the neural thread protein (AD7C-NTP) (developed by Nymox) demonstrated higher levels in urine from AD patients than from non-AD dementia patients or healthy controls. However, the mean levels were significantly lower in early AD cases, suggesting the test is not reliable for testing for early onset of AD.
No biochemical method is as yet suited for the firm diagnosis of early stages of AD, rather they merely help to confirm the clinical diagnosis of advanced cases. Clearly, more advanced techniques are needed to allow early diagnosis before onset of clinical symptoms that signal irreversible brain damage.
Finally, not only for diagnostic purposes but also in e.g. pre-clinical research and development, A-beta binding molecules are useful as research tools. Widely used are the antibodies already mentioned above, i.e. antibody 3D6 and antibody m266. Antibody 3D6 binds to A-beta with a relatively low affinity and may therefore not be suitable for all purposes. Antibody m266 cross-reacts with N-terminally truncated versions of A-beta, such as p3, which does not allow to distinguish between disease-relevant A-beta species, such as A-beta(1-40) and A-beta(1-42), and other molecules such as p3.
In view of the above, it is an object of the invention to provide pharmacologically active agents, as well as compositions comprising the same, that can be used in the prevention, treatment, alleviation and/or diagnosis of diseases, disorders or conditions associated with A-beta and/or mediated by A-beta, such as AD, and to provide methods for the prevention, treatment, alleviation and/or diagnosis of such diseases, disorders or conditions, involving the use and/or administration of such agents and compositions. Such agents may also be useful for doing research into the field of AD in general and, specifically, into the elucidation of AD disease mechanisms and potential therapeutic and/or prophylactic mechanisms.
In particular, it is an object of the invention to provide such pharmacologically active agents, compositions and/or methods that provide certain advantages compared to the agents, compositions and/or methods currently used and/or known in the art. These advantages include improved therapeutic and/or pharmacological properties and/or other advantageous properties (such as, for example, improved ease of preparation and/or reduced costs of goods), especially as compared to conventional antibodies against A-beta or fragments thereof as those described in the above section. Further advantages will become clear from the further description below.
More in particular, it is an object of the invention to provide novel A-beta binding molecules and, specifically, polypeptides binding to mammalian and, especially, human A-beta, wherein such molecules or polypeptides are suitable for the above diagnostic, therapeutic and research purposes.